Avian influenza (AI) is a serious pathogen that infects avians, other animals, and humans. Since 1997, there have been several incidents of transmission of AI virus to humans (Subbarao et al., 1998; Ungchusak et al., 2005). Evidence also shows that genetic recombination between avian and human influenza viruses have occurred on multiple occasions in medical history (Kawaoka et al., 1989). Since avians and humans are in close contact, it is believed that the generation of new AI virus strains that could potentially cross the species barrier into the human population will continue to be a public health concern.
Mass vaccination of avians appears to be the most promising approach to prevent dissemination of AI virus and to reduce the risk of human pandemics. Vaccination of avians with inactivated whole virus vaccines has been performed in some countries over the past several years. These AI vaccines are prepared from amnio-allantoic fluid harvested from infected eggs, and are subsequently inactivated by formalin or β-propiolactone (Tollis and Di Trani, 2002). However, the unpredictable emergence of new AI virus strains, the evolution of Al virus into a form highly lethal to chicken embryos (Wood et al., 2002), and possible dissemination of lethal AI strains by bioterrorists make the rapid development and timely supply of safe and efficacious AI vaccine a crucial, yet very difficult, task. In addition, it is not possible to discriminate field-infected chickens from those previously vaccinated with inactivated AI viruses of the same strains (Normile, 2004).
An experimental recombinant fowlpox virus encoding the hemagglutinin (HA) of an AI virus has protected chickens against a H5N2 AI virus challenge after wing-web puncture, although the hemagglutination-inhibition (HI) serologic response was negligible (Beard et al., 1992). Chickens inoculated through the wing web with a live recombinant vaccinia virus expressing HA also developed protective immunity against a lethal AI virus challenge with low levels of serum HI antibody detected (Chambers et al., 1988). Although AI isolates of waterfowl-origin that have a tropism for the alimentary tract have been inoculated into chickens as an oral AI vaccine (Crawford et al., 1998), those isolates are not expected to be broadly effective against new AI virus strains, due to the inherently dynamic evolution of this type of virus.
Avians have also been immunized by subcutaneous injection of HA proteins expressed from baculovirus vectors (Crawford et al., 1999), and inoculation of an expression plasmid encoding HA into the skin using a gene gun (Fynan et al., 1993). These AI vaccines are able to protect avians from exhibiting clinical signs and death, and reduce respiratory and intestinal replication of a challenge virus containing homologous HA. There is also evidence that a low-cost aerosol AI vaccine expressing HA from a Newcastle disease virus vector (Swayne, 2003) or a recombinant influenza virus containing a non-pathogenic influenza virus backbone may be efficacious (Lee et al., 2004; Webby et al., 2004).
Most of the above AI vaccines rely upon labor-intensive parenteral delivery. The oral and aerosol AI vaccines suffer from inconsistencies in delivering a uniform dose to individual birds during mass-inoculation. The replicating vectors used in some vaccines also pose a biohazard by introducing unnatural microbial forms to the environment. The recombined influenza virus vaccine could even generate harmful reassortments through recombination between a reassortant influenza virus and a wild AI virus concurrently circulating in the environment (Hilleman, 2002).
There are several noteworthy reasons for utilizing recombinant Adenovirus (“Ad”) vectors as a vaccine carrier. Ad vectors are able to transduce both mitotic and postmitotic cells in situ. Additionally, preparation of Ad stocks containing high titers of virus (i.e., greater than 1012 pfu [plaque-forming units] per ml) are easy to generate, which makes it possible to transduce cells in situ at high multiplicity of infection (MOI). Ad vectors also have a proven safety record, based on their long-term use as a vaccine. Further, the Ad virus is capable of inducing high levels of gene expression (at least as an initial burst), and replication-defective Ad vectors can be easily bioengineered, manufactured, and stored using techniques well known in the art.
Ad-based vaccines are more potent than DNA vaccines due to Ad vector's high affinity for specific receptors and its ability to escape the endosomal pathway (Curiel, 1994). Ad vectors may transduce part of a chicken embryo through binding of its fiber to the coxsackie and adenovirus receptor (CAR) found on the surface of chicken cells (Tan et al., 2001). In addition, at least one of the Ad components, hexon, is highly immunogenic and can confer adjuvant activity to exogenous antigens (Molinier-Frenkel et al., 2002).
Ad-based vaccines mimic the effects of natural infections in their ability to induce major histocompatibility complex (MEW) class I restricted T-cell responses, yet eliminate the possibility of reversion back to virulence because only a subfragment of the pathogen's genome is expressed from the vector. This “selective expression” may solve the problem of differentiating vaccinated-but-uninfected animals from their infected counterparts, because the specific markers of the pathogen not encoded by the vector can be used to discriminate the two events. Notably, propagation of the pathogen is not required for generating vectored vaccines because the relevant antigen genes can be amplified and cloned directly from field samples (Rajakumar et al., 1990). This is particularly important for production of highly virulent AI strains, such as H5N1, because this strain is too dangerous and difficult to propagate (Wood et al., 2002). In addition to the above criteria, commercial concerns factor heavily in the poultry industry. The current AI vaccine alone costs about 7 cents per bird, not counting the labor of injecting running birds (Normile, 2004).
Replication-incompetent E1/E3-defective human Ad serotype 5 (Ad5)-derived vectors have been extensively studied in mammals (Graham and Prevec, 1995). Although chickens have been immunized by subcutaneous or intradermal injection of an avian Ad chicken embryo lethal orphan (CELO) viral vector encoding an antigen (Francois et al., 2004), the CELO vector has a low compliance rate and could be potentially harmful due to its ability to replicate in chicken cells. Since CELO possesses no identifiable E1, E3, and E4 regions (Chiocca et al., 1996), a replication-incompetent CELO vector is not available as a carrier for immunization at this time. The present invention addresses this need by providing a safe and efficient method for gene delivery to protect avians in a wide variety of disease settings, and consequently prevent transmission of avian pathogens to humans.